Published online Aug 28, 2015. doi: 10.3748/wjg.v21.i32.9566
Peer-review started: March 31, 2015
First decision: April 23, 2015
Revised: May 15, 2015
Accepted: July 8, 2015
Article in press: July 9, 2015
Published online: August 28, 2015
AIM: To investigate the effect of carvedilol on angiogenesis and the underlying signaling pathways.
METHODS: The effect of carvedilol on angiogenesis was examined using a human umbilical vascular endothelial cell (HUVEC) model. The effect of carvedilol on cell viability was measured by CCK8 assay. Flow cytometry was used to assess the effect of carvedilol on cell cycle progression. Cell migration, transwell migration and tube formation assays were performed to analyze the effect of carvedilol on HUVEC function. Vascular endothelial growth factor (VEGF) induced activation of HUVECs, which were pretreated with different carvedilol concentrations or none. Western blot analysis detected the phosphorylation levels of three cell signaling pathway proteins, VEGFR-2, Src, and extracellular signal-regulated kinase (ERK). The specific Src inhibitor PP2 was used to assess the role of Src in the VEGF-induced angiogenic pathway.
RESULTS: Carvedilol inhibited HUVEC proliferation in a dose-dependent manner (IC50 = 38.5 mmol/L). The distribution of cells in the S phase decreased from 43.6% to 37.2%, 35.6% and 17.8% by 1, 5 and 10 μmol/L carvedilol for 24 h, respectively. Carvedilol (10 μmol/L) reduced VEGF-induced HUVEC migration from 67.54 ± 7.83 to 37.11 ± 3.533 (P < 0.001). Carvedilol concentrations of 5 μmol/L and 10 μmol/L reduced cell invasion from 196.3% ± 18.76% to 114.0% ± 12.20% and 51.68% ± 8.28%, respectively. VEGF-induced tube formation was also reduced significantly by 5 μmol/L and 10 μmol/L carvedilol from 286.0 ± 36.72 to 135.7 ± 18.13 (P < 0.05) and 80.27 ± 11.16 (P < 0.01) respectively. We investigated several intracellular protein levels to determine the reason for these reductions. Treatment with 10 μmol/L carvedilol reduced VEGF-induced tyrosine phosphorylation of VEGFR-2 from 175.5% ± 8.54% to 52.67% ± 5.33% (P < 0.01). Additionally, 10 μmol/L carvedilol reduced VEGF-induced ERK 1/2 phosphorylation from 181.9% ± 18.61% to 56.45% ± 7.64% (P < 0.01). The VEGF-induced increase in Src kinase activity was alleviated by carvedilol [decreased from 141.8% ± 15.37% to 53.57 ± 7.18% (P < 0.01) and 47.04% ± 9.74% (P < 0.01) at concentrations of 5 and 10 μmol/L, respectively]. Pretreatment of HUVECs with Src kinase inhibitor almost completely prevented the VEGF-induced ERK upregulation [decreased from 213.2% ± 27.68% to 90.96% ± 17.16% (P < 0.01)].
CONCLUSION: Carvedilol has an anti-angiogenic effect on HUVECs. This inhibitory effect is mediated by VEGF-induced Src-ERK signaling pathways.
Core tip: Carvedilol has been used for the treatment of portal hypertension for many years. In this study, carvedilol directly inhibited the proliferation and tube formation of cultured human umbilical vascular endothelial cells. Moreover, this study is the first to investigate the mechanism of the anti-angiogenic effect of carvedilol, which functions by inhibiting vascular endothelial growth factor-induced mitogen-activated protein kinase signaling pathways. These novel activities of carvedilol provide insight into the anti-angiogenic mechanisms involved, and highlight its potential therapeutic application against angiogenesis-dependent liver fibrosis.